PROJECT SUMMARY Genetic screens in mice and the naturally occurring genetic variation in humans have provided a valuable resource to identify genes implicated in hair cell function and deafness. Two examples is the identification of genes involved in Usher syndrome, the most common form of deaf-blindness, and the role of Myh9 in both syndromic and non-syndromic deafness. We have used the power of Drosophila genetics to identify new genes involved in hearing and deafness. The auditory organs of Drosophila and vertebrates have a number of molecular and functional similarities despite being widely separated in evolutionary time. We identified mutations in Ubr3, an E3 ubiquitin ligase, that cause a physical detachment of the sensory components of Johnston's organ from the fly antenna. Strikingly, this phenotype is identical to that seen in mutations in Drosophila Myosin VIIa. Since Myosin VIIa mutations in humans cause Usher Syndrome type IB, it is possible that Ubr3 may regulate Myosin VIIa function in invertebrates and vertebrates. Our data suggest that Ubr3 genetically interacts with Myosin VIIa and Ubr3 and Myosin VIIa physically and genetically interact with Drosophila homologues of two other Usher syndrome proteins, PCDH15 and Sans. However, we have found that Ubr3 does not modify Myosin VIIa, but instead mono-ubiquitinates non-muscle Myosin II. This modification increase an interaction between the two myosins, and fine-tuning the level of this interaction appears critical for Myosin VIIa function. In the present proposal, we will expand on the use of Drosophila as a model system to understand deafness by characterizing the roles of Ubr3, Myosin II and Myosin VIIa in hearing in Drosophila (Aim 1). We will then test the function of Ubr3 in the development and function of mouse hair cells, and will test whether the interaction of Myosin II and Myosin VIIa is conserved in mice (Aim 2). Finally, we will carry out a genetic screen of 3000 Drosophila genes that may be involved in human disease using newly developed protein knockdown technology to identify genes that play a role in hearing in Drosophila (Aim 3).